Rev. Int. Contam. Ambient. 11 ( l ) , 9-12, 1995

INDUCTION OF MICRONUCLEI IN PERIPHERAL ERYTHROCYTES OF CYPRINUS CARPIO FISH BY METHYL PARATHION

Júlio César NEPOMUCENO and Mário Antonio S P A N ~

Universidade Federal de Uberlandia. Departamento de Biociencias, Bloco D, Carnpus Umuarama. CEP 38.400- 902 Uberlandia (MG) , Brasil

(Recibido mayo 1994, aceptado febrero 1995)

Keywords: Cypinus c a p , micronucleus test, methyl parathion

ABSTRACT

Methyl parathion (MP) is a commonly used organophosphorus insecticide, commercially available as Folidol 600. This chemical substance was tested to determine micronucleus (MN) frequencies in peripheral erythrocytes of the fish Cypnnus c e i o treated in vivo. The concentrations tested were 1.25,2.50,5.00 and 7.50 ppm. Fish were killed after 24 or 48 h of treatment. There was a significant increase in the frequency of micronuclei in the animals treated with 2.50 and 5.00 ppm after 24 h. At higher dosages this effect disap peared probably due to the increased cell lethality. The frequency of MN induction was lower at 48 than at 24 h after treatment. There was a dosedependent increase in MN fre- quency which, however, was significant only at the highest concentration (7.50 ppm) compared to control.

RESUMEN

El metil paratión (MP) es un insecticida organofosforado disponible comercialmente como Folidol 600. Esta substancia fue probada para determinar la frecuencia de micronúcleos (MN) en eritrocitos periféricos de peces Cyprinus ca+o tratados in vivo. Las concen- traciones utilizadas fueron 1.25,2.50, 5.00 y 7.50 ppm. Los peces fueron sacrificados 24 y 48 h después del tratamiento. Se notó un aumento significativo en la frecuencia de mi- cronúcleos en los animales tratados con 2.50 y 5.00 ppm y sacrificados después de 24 h. A concentraciones mayores este efecto desapareció probablemente debido al incremento de la letalidad celular. La frecuencia de inducción de MN fue menor después de 48 h que a 24 h del tratamiento. En la primera hubo una elevación en la frecuencia de MN de- pendiente de la concentración que, sin embargo, fue significativa cuando se comparó con el testigo solamente en la mayor (7.50 ppm).

INTRODUCTION

Methyl parathion (MP) is a broad-spectrum anticholines- terase organophosphorus insecticide widely used against crop pests. In Brazil it is also being employed at piscicul- ture sites to select the zooplankton and to eliminate pre- dators. Methyl parathion is commercially available as Foli- do1 600.

Negative results have been reported in mutagenic assays with MP on Salmonella and Streptomyces (Carere et al. 1978), and positive results in Salm¿rneIla with and without S9 mix (Waters et al. 1980, Rashid and Mumma 1984, Breau et al. 1985). The compound is probably mutagenic

in Escha'chia coli, inducing 5-methyltryptophan (5-MT) resistance (Mohn 1973a, b). MP is mutagenic in somatic and germ line cells of Drosophila and induces sex-linked recessive lethals in immature maie germ cells (Tripathy d al. 1987).

The results from studies on mammais are also conflict- ing. Negative results were obtained for chromosome abe- rrations in bone marrow ceils and spermatogonia, and for the frequency of pre and postimplantation fetal lethality in a dominant lethal mutation assay in mice (Degraeve and Moutschen 1984). MP caused no observable chromo- some damage either in human cells in uitro or in mouse cells in vivo (Huang 1973). However, an increased fre-

J.C. Nepomuceno and M.A. Spanó

quency of chromosome aberrations was described in mou- se bone marrow cells (Kuinnyi 1975) and of sister chroma- tid exchanges in human lymphoid cells (Sobti ei al. 1982) and in V79 Chinese harnster ovary cells (Chen d al. 1981).

The micronucleus test (MNT) was developed in marn- malian polychromatic erythrocytes of the bone marrow (Schmid 1975). Nevertheless, it has also been applied to nucleated piscine erythrocytes in the genotoxicity evalua- tion of test compounds in vivo (Hooftman and de Raat 1982, Hose et al. 1984, Manna d al. 1985, 1987, Das and Nanda 1986). This assay can detect agents which cause chromosome breaks and agents which affect the mitotic spindle. Thus, micronuclei (MN) are indicators of clasto- genic or aneugenic effects.

Since micronucleated piscine erythrocytes have been proved to be sensitive indicators of genetic damage, the purpose of our study was to evaluate the cytogenetic (clas- togenic or aneugenic) effects of methyl parathion in Cy- pznus carpo fish using the MNT.

MATERLALS AND METHODS

Chemicai agent

Methyl parathion (0,Odimethyl-0pnitrophenyl phosphoro- thioate) (CAS NQ 2980(M) is an organophosphoms insec- ticide commercially available as Folidol 600 (Bayer). The chemical structure of the compound is shown in figure 1.

MP was dissolved in water collected from an IBAMA pisciculture site and diluted to the desired concentrations. C@'nus carpa fish were exposed to methyl parathion con- taminated water for 24 and 48 h.

Zn vivo assay on peripheral erytbrocytes of Cyprinus car- pio for the detennhtion of micronucleus frequencies

Cyprinus carpio fish weighing approximately 1.0 g were purchased from an IBAMA pisciculture site, Uberlbdia (MG) Brazil, and kept in laboratory aquaria containing water collected from the same site, in order to maintain zooplankton concentration and avoid chlorinated tap wa- ter. The aquaria were aerated frequentiy and the animals

Fíg. 1. Strucairal formula of the merhyl parathion

were allowed to acclimatize for one week prior to treat- ment. A batch of 56 fish were equally released in four dif- ferent aquaria (fourteen animals in each aquarium), con- taining different concentrations (1.25, 2.50, 5.00 or 7.50 ppm) of MP dissolved in the aquarium water. Fish were then killed, following 24 and 48 h of treatment. These time periods were arbitrfly chosen. Fourteen fish kept in the same kind of water, but without MP, and killed at the same time intervals served as controls.

Seven fish were used for each concentration level and for each sampling period, including the controls. Fish were cut in the caudal region, and blood smears were im- mediately prepared and fixed in absolute methanol for 15 min. On the following day, the material was stained with Giemsa diluted 1:20 in sodium phosphate buffer, pH 6.8, for 10 minutes. A total of 2,000 cells, 500 per slide, from each individual were examined microscopically un- der 1000 X magnification. Coded and randomized slides were scored using blind review by a single observer. The number of micronuclei (MN) and micronucleated cells (MNC) were expressed per 100 ceils.

The data were analyzed statistically by the conditional test for the detection of rare events (Pereira 1991), with the level of significance set at a = 0.05.

RESULTS AND DISCUSSION

The frequencies of MN and MNC observed in Cyprinw carpo treated with MP for 24 h were significantly higher in fish treated with 2.50 and 5.00 pprn (P < 0.008 and 0.005, respectively) than those in control fish. At a concentration of 7.50 ppm, the increase in frequency was not significant (P > 0.07), probably due to the increased cell lethality (Ta- ble 1). An inconsistent result was found in this experi- ment. The frequency of MN and MNC observed in fish treated with 1.25 pprn of MP for 24 h was significantly l e wer than that in controls (P < 0.005) (Table 1).

High frequencies of MN and MNC were expected to be found in the controls, based on previous observations of high incidence of MN in fish (Oreochromis mossambiczls) collected from the same site of pisciculture (IBAMA- Uberlhdia) due to environmental pollutants (Nepomu- ceno and Spanó 1992).

On the other hand, the frequencies of MN and MNC present in C y p i n w capo treated with MP for 48 h were significantiy higher (P < 0.005) only in fish treated with 7.50 pprn (Table 11). For 1.25, 2.50 and 5.00 pprn doses, the data on MN and MNC did not differ significantly from the respective controls (P > 0.4, 0.08 and 0.06, res- pectively) (Table U). The lower frequencies of micronu- clei obtained after 48 h of treatment compared to those obtained after 24 h maybe due to the toxicity of MP leading to cell death.

MICRONUCLEI IN FISH ERYTHROCYTES BY METHYL PARATHION

TABLE 1. FREQUENCY O F MICRONUCLEATED CELLS (MNC) AND MICRONUCLEI (MN) IN PERIPHERAL ERYIHROCYTES O F CYPRINUS CARPIO FISH EXPOSED T O METHYL PARATHION CONTAMINATED WATER

FOR 24 HOURS

Dose No. of Total Total Total %(%) I SD

(pprn) indiv. ceiis MN MNC MN MNC

0.00 7 14000 72 7 1 0.51 I 0.35 0.50 + 0.35

1.25 7 14000 41* 41* 0.29 I 0.20 0.29 + 0.20

2.50 7 14000 105* 102* 0.75 I 0.32 0.72 I 0.30

5.00 7 14000 278* 275* 1.98 I 2.58 1.96 I 2.55

7.50 7 14000 9 1 9 1 0.65 * 0.57 0.65 ? 0.57

* Significantly different from the control according to the conditional test for the detection of rare events (Pereira 1991) with the leve1 of significancr set at a = 0.05.

Most of the earlier studies with organophosphates used bacteria1 test systems and the reports on mammalian test systems were mostly negative possibly due to the toxi- city and the rapid degradiation of the test chemicals in in riirio and in vitro experiments (Wild 1975).

The induction of 5-MT resistance mutations in E. coli shows that MP is probably mutagenic. However, under the experimental conditions descnbed, the compound is not significantly mutagenic at doses which do not inacti- vate the cells, whereas at higher doses an increase in rela- tive mutation frequency occurs with a parallel decrease in survival (Mohn 1973a).

Incubation of human lymphoid cells (M-007) with 20 p/ml caused a significant increase in SCEs. However, cytotoxic effects of MP were dose related and led to cell death (Sobti et al. 1982).

Wild (1975) described the general aspects of the che- mistry, toxicity and metabolism of organophosphates. According to Breau et al. (1985), the most mutagenic phos- phorothioates are those containing strong electron-with- drawing substitutes (as in MP) .

Kurinnyi (1975) observed chromosome lesions in bo- ne marrow of mice treated with 10 mg/kg MP, consider- ing this compound to be slightly clastogenic.

There is a lack of information about the formation and frequency of micronuclei in the haeinatopoietic tissues of Cypnnus carpio fish. There is no information about the time taken for the completion of the mitotic cycle in blood cells, nor about the time taken for the blood cells to reach the peripheral circulation. Because of this, we cannot assure that micronuclei found had been formed during haematopoiesis at different times (24 or 48 h) of

TABLE 11. FREQUENCY O F MICRONUCLEATED CELLS (MNC) AND MICRONUCLEI (MN) IN PERIPHERAL ERYTHROCYTES O F CYPRINUS CARPIO FISH EXPOSED T O METHYL PARATHION CONTAMINATED WATER

FOR 48 HOURS

Dose No. of Total Total Total x (%) I SD

( P P ~ ) indiv. ceiis MN MNC MN MNC

7.50 7 14000 168* 168* 1.20 I 1.85 1.20 I 1.85

* Significantly different from the control according to the conditional test for the detection of rare wenu (Pereira 1991) with the leve1 of significance set at a = 0.05.

J.C. Nepomuceno and MA. Spanó

treaunent with MP. However, in spite of these considera- tions, based on our data and on previous reports, we may conclude that MP has genotoxic effects.

Although organophosphates are not persistent in the environment and do not pose a serious residue problem (Wild 1975), the continued and increased use of these pesticides may lead to unintentional deleterious effects on the environment and on animal and human health.

ACKNOWLEDGMENTS

We are grateful to IBAMA (Uberlhdia, MGBrasil) which provided the animais for this study. This work was suppor- ted by Universidade Federal de Uberlhdia and CNPq. The authors are aiso indebted to two anonyrnous referees for criticai reading of the original manuscript.

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